Abstract: The objective of this proposal is to elucidate brainstem regulatory cholinergic circuitries involved in the vagus nerve-based inflammatory reflex during sepsis. Sepsis is a complex lethal condition defined as ?life- threatening organ dysfunction caused by a dysregulated host response to infection?. The incidence of sepsis is increasing and costs approx $17B per year. Moreover, many ?sepsis survivors? develop immune dysfunction, chronic illness, and severe functional disabilities, which may result in death. An essential understudied aspect of sepsis is the involvement of the nervous system. We have pioneered studies on the role of the nervous system and the inflammatory reflex and have provided important insight related to the role of this neural immunomodulatory mechanism in sepsis and other conditions characterized by immune dysfunction. These preclinical findings recently led to successful clinical exploration of the inflammatory reflex via vagus nerve stimulation in patients with rheumatoid arthritis. Despite this progress, there are major gaps in our understanding of the vagus nerve-based inflammatory reflex and its relevance to sepsis. Efferent vagus nerve fibers originate in two brainstem nuclei, the dorsal motor nucleus of the vagus (DMN) and nucleus ambiguus (NA). Experimental evidence supports that efferent vagus nerve fibers interact with the splenic nerve in ganglia of the celiac (solar) plexus within the inflammatory reflex. However, the brainstem origin of the efferent vagus nerve fibers critical to the inflammatory reflex and the specific role(s) of these circuitries in regulating immune and metabolic indices during sepsis are presently unknown. We hypothesize that brainstem DMN and NA efferent cholinergic fibers have differential roles in the inflammatory reflex during sepsis. This hypothesis will be tested by the following Specific Aims: Specific Aim 1. Elucidate the specific role of brainstem cholinergic circuitries in the vagus nerve-based inflammatory reflex. Working hypothesis: Efferent vagus nerve fibers originating in the brainstem DMN and NA differentially contribute to the vagus nerve-based inflammatory reflex to the spleen. We will utilize: 1) recombinant adeno-associated viruses transcriptionally activated by Cre recombinase (Cre-on) to achieve precise molecular mapping of brainstem cholinergic circuitries in mice; 2) selective optogenetic cholinergic stimulation of brainstem circuitries to examine acetylcholine release in the spleen and anti-inflammatory effects in endotoxemic mice; and 3) selective vagotomies to confirm the role of the vagus nerve. Specific Aim 2. Evaluate the effects of selective cholinergic activation of brainstem nuclei on inflammatory and metabolic markers and survival following sepsis. Working hypothesis: Selective cholinergic optogenetic stimulation of DMN and NA differentially alleviates sepsis derangements. We will perform experiments using selective optogenetic stimulation of brainstem nuclei of mice following CLP to examine effects on: 1) inflammatory and metabolic indices in acute and chronic stages; and 2) survival.